Summary of Work: This project applies biochemical and molecular biological approaches to the study of the complex interplay of the molecular signaling events that occur within T cells in response to engagement of the T cell antigen receptor (TCR) and co-stimulatory receptors. It is anticipated that a better understanding of the events that occur in the normal course of T cell activation will provide logical targets to probe with regard to their importance in immune pathogenicities, such as those characterized by immunosenescence in aged animals. The current focus is upon the kinases and phosphatases that regulate the phosphorylation status of key proteinaceous and phosphoinositide-based signaling molecules. Of particular interest has been the TCR-proximal protein tyrosine kinase (PTK) ZAP-70. By comparing the signaling pathways in T cells that are either deficient or replete for ZAP-70 we have found that ZAP-70 regulates the activity of the transcription factor NF-kappa-B, which is involved in increasing gene expression at key loci, such as that of the growth and survival factor, interleukin 2. Using a similar approach with SLP-76-negative T cells, we found that the cytosolic adapter protein and ZAP-70 substrate, SLP-76 is also required for NF-kappa-B activation. Further analysis has established that ZAP-70 and SLP-76 are required for the activation of PKC-theta, a required event in NF-kappa-B activation. Additional investigation into the role of ZAP-70 in mediating TCR signaling has shown that partial TCR signaling in response to maximal engagement of the TCR occurs in the absence of ZAP-70 expression. This pathway is likely to play an important role in signaling for the establishment and maintenance of tolerance/anergy. Additional studies related to this project have investigated the role of the lipid phosphatase PTEN in regulating growth, proliferation and activation of T cells. This phosphatase acts in opposition to the lipid kinase, PI3K. Previously our laboratory demonstrated that unlike normal T cells, the leukemic Jurkat T cell line fails to express PTEN. To explore the importance of this phosphatase in T cell function and response to TCR stimulation we have created a Jurkat T cell line that expresses PTEN under a tightly-controlled expression system. Notably, Jurkat T cells expressing PTEN seem to have little increase in susceptibility to apoptosis, but rather are smaller in size and proliferate more slowly than PTEN-negative cells. We propose from these studies that the net balance of activity between PTEN and PI3K plays a pivotal role in the progression from quiescence to the profound replication rate associated with T cell activation. Unregulated changes in this balance, as characterized by the loss of PTEN expression likely predispose towards the development of leukemia. Whether or not more subtle perturbations of the pathways regulated by these enzymes are involved in the reduced proliferative responsiveness of immunosenescent T cells remains to be established.